This proposal aims to develop and test the gene transfer efficiency of chemically defined Reductively Activated Melittin PEGylated glycoproteins (RAMPs). RAMPs will be prepared on solid support by joining a melittin peptide, PEG-peptide and glycopeptides by disulfide bond formation. The resulting RAMPs will bind to DNA to form polyplexes that target hepatocytes in vivo and undergo a glutathione triggered release of melittin in endosomes. This proposal is innovative in its development of a means to systematically vary the composition, sequence and reductive stability of homogeneous RAMPs to control the intracellular location and release rate of melittin to improve the level of gene expression. The proposed solid phase synthesis of RAMPS is based on our previous success in developing reductively activated peptides containing terminal Cys residues that mediate a triggering release of DNA in the intracellular reducing environment, dramatically increasing gene transfer efficiency. Since this initial discovery, we have developed Cys-terminated polyethylene glycol (PEG) - peptides, and glycopeptides containing a natural triantennary N-glycan asialoglycoprotein receptor ligand. We recently developed Cys-terminated melittin peptides as potent in vitro gene transfer peptides. These advances resulted in the development of Reductively Activated Melittin PEGylated glycoproteins (RAMPs) by a random co-polymerizing of Cys terminated PEG-peptide, glycopeptides and melittin. Randomly co-polymerized RAMP polyplexes mediated specific targeting to heptocytes in mice without observable toxicity and demonstrated significant luciferase expression from a 5 g DNA dose delivered via the tail vein. However, the gene transfer efficiency of randomly co-polymerized RAMP polyplexes can still be significantly improved by precisely controlling the sequence, composition and the intracellular release location of melittin. To enhance the gene transfer efficiency of RAMPs, we propose to develop homogenous RAMPs of precise sequence and composition using a novel solid synthesis. These will be used to test the hypothesis that delaying the release of melittin until the late endosomes by incorporating penicillamine disulfide bonds will enhance gene transfer efficiency. We propose to test RAMP polyplexes of defined sequence, composition and stability for in vitro gene transfer activity in primary mouse hepatocytes. Potent RAMP polyplexes will be used to deliver luciferase expressing plasmids to hepatocytes in vivo. Quantitative bioluminescence imaging (BLI) will be used to determine the efficiency of RAMP polyplexes relative to an equivalent hydrodynamic dose of DNA. We propose to evaluate the liver toxicity of RAMP polyplexes compared to hydrodynamic dosing. RAMP polyplexes will be used to treat Factor VIII deficiency in hemophilic mice. The correction of this coagulation defect in hemophilic mice will be used to establish the therapeutic potential of RAMP polyplexes. The results of this study aim to develop the first homogenous reductively activated nonviral gene delivery carrier to achieve comparable efficiency as hydrodynamic dosing, using much smaller dosing volumes and resulting in less liver toxicity. PUBLIC HEALTH RELEVANCE: This proposal aims to develop and test homogeneous Reductively Activated Melittin PEGylated glycoproteins (RAMPs) as a new class of gene delivery agents that improve the delivery and expression of DNA in animals.